GSM4890933: AR ChIP MED19-LNCaP CS media 2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
AR
Cell type
Cell type Class
Prostate
Cell type
LNCAP
Primary Tissue
Prostate
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
LNCaP
antibody
AR 441 mouse monoclonal, Santa Cruz, cat # sc-7305) and (AR D6F11 rabbit monoclonal, Cell Signaling Technology, cat # 5153)
treatment
EtOH vehicle 4 h
phenotype
MED19 overexpression
disease
prostate cancer
cell line
LNCaP
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cell pellets were resuspended in nuclei isolation buffer (50 mM Tris–pH 8.0, 60 mM KCl, 0.5% NP40), nuclei collected, and resuspended in sonication buffer (RIPA buffer). Samples were sonicated in TPX PMP tubes (Diagenode) for 60 min (30 sec. on, 30 sec. off) in a Bioruptor sonicator (Diagenode). Inputs (10%) were collected and supernatants were then incubated overnight with the following antibodies pre-incubated with Protein A and Protein G Dynabeads (Invitrogen): a mixture of AR antibodies (AR 441 mouse monoclonal, Santa Cruz Biotechnology, cat # sc-7305) and (AR D6F11 rabbit monoclonal, Cell Signaling Technology, cat # 5153); DYKDDDDK Tag (FLAG epitope) (Cell Signaling Technology, cat # 14793); or H3K27 acetylation (Active Motif, cat # 39034). Control ChIPs were performed with normal mouse IgG (Santa Cruz Biotechnology, cat # sc-2025) and normal rabbit IgG (Sigma Aldrich, cat # 12-370). Immunocomplexes were then washed and cross-linking reversed overnight at 65 °C with 5 M NaCl. DNA was isolated with the Zymo Chip DNA Clean and Concentrator kit (Zymo Research). libraries were prepared according to the protocol described in PMID: 30679424. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from a 10% polyacrylamide gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Sequencing was performed using Illumina HiSeq4000 Sequencing (HiSeq 4000 Single Read 50 Cycle Lane).